SUMMARY Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling.

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate to distal cilia. This triggers otherwise forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. Whilst cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Visualization of signal transduction within live primary cilia constitutes a technical challenge due to its sub-micron dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, sensitivity and wide dynamic range.

Primary cilia function as specialized compartments for signal transduction. The stereotyped structure and signaling function of cilia inextricably depend on the selective segregation of molecules in cilia. However, the fundamental principles governing the access of soluble proteins to primary cilia remain unresolved. We developed a methodology termed Chemically-Inducible Diffusion Trap at Cilia (C-IDTc) to visualize the diffusion process of a series of fluorescent proteins ranging in size from 3.2 to 7.9 nm into primary cilia. We found that the interior of the cilium was accessible to proteins as large as 7.9 nm. The kinetics of ciliary accumulation of this panel of proteins was exponentially limited by their Stokes radii. Quantitative modeling suggests that the diffusion barrier operates as a molecular sieve at the base of cilia. Our study presents a set of powerful, generally applicable tools for the quantitative monitoring of ciliary protein diffusion under both physiological and pathological conditions.

tional experimentation following the publication of the paper has raised an issue with the results reported in Figure S2. The paper reported that disassembly of primary cilia can occur through ''decapitation'' of ciliary tips to release vesicles containing ciliary material. The phosphoinositide 5-phosphatase Inpp5e restricts the process. In Figure S2, the authors presented data supporting this role, showing that increased expression of Inpp5e promoted the suppressive effect and that the enzyme's catalytic activity was required. After publication, the authors discovered errors in the plasmids encoding 5HT 6-YFP-Inpp5e(WT)and 5HT 6-YFP-Inpp5e(PD, phosphatase dead), arising from the published DNA sequence of the parent plasmid, that resulted in expression plasmids with a frameshift between YFP and Inpp5e. They have repeated the same experiments with corrected constructs (available through Addgene-#96808, 96809), and obtained results consistent with the conclusions originally presented. With this note, we and the authors would like to alert the community and direct readers to additional data related to Figure S2, which have been deposited in Mendeley Data and can be accessed at https://data.mendeley.com/datasets/5vnt9wsbwf/1. The remaining findings in the paper are unchanged, and the new data do not alter the main conclusions of the paper. Additionally, the authors have informed us that they failed to list Sté phane Schurmans as an author despite a critical contribution to the work through generation of the Inpp5e-knockout MEFs that enabled collection of the data shown in Figures 1 and 3. To correct this oversight, we have added Sté phane Schurmans as an author. All co-authors have approved this addition and the corrected author list is shown below.

The primary cilium is a solitary hair-like organelle on the cell surface that serves as an antenna sensing ever-changing environmental conditions. In this review, we will first recapitulate the molecular basis of the polymodal sensory function of the primary cilia, specifically focusing on transient receptor potential (TRP) channels that accumulate inside the organelle and conduct calcium ions (Ca2+). Each subfamily member, namely TRPP2 TRPP3, TRPC1 and TRPV4, is gated by multiple environmental factors, including chemical (receptor ligands, intracellular second messengers such as Ca2+), mechanical (fluid shear stress, hypo-osmotic swelling), or physical (temperature, voltage) stimuli. Both activity and heterodimer compositions of the TRP channels may be dynamically regulated for precise tuning to the varying dynamic ranges of the individual input stimuli. We will thus discuss the potential regulation of TRP channels by local second messengers. Despite its reported importance in embryonic patterning and tissue morphogenesis, the precise functional significance of the downstream Ca2+ signals of the TRP channels remains unknown. We will close our review by featuring recent technological advances in visualizing and analyzing signal transduction inside the primary cilia, together with current perspectives illuminating the functional significance of intraciliary Ca2+ signals.

Chemically-inducible rapid manipulation of small GTPase activity has proven a powerful approach to dissect complex spatio-temporal signaling of these molecular switches. However, overexpression of these synthetic molecular probes freely in the cytosol often results in elevated background activity before chemical induction, which perturbs the cellular basal state and thereby limits their wide application. As a fundamental solution, we have rationally designed and newly developed a strategy to remove unwanted background activity without compromising the extent of induced activation. By exploiting interaction between a membrane lipid and its binding protein, target proteins were translocated from one organelle to another on a time scale of seconds. This improved strategy now allows for rapid manipulation of small GTPases under a physiological state, thus enabling fine dissection of sophisticated signaling processes shaped by these molecules.

Organs such as the lung and the kidney are composed of epithelial and endothelial tubule-forming networks. To engineer such organs, it would be desirable to control the shape, spatial orientation and interconnectedness of the forming tubules. To study this, channels were formed in extracellular matrix (ECM) gels and were subsequently filled with Madin-Darby canine kidney epithelial cells or human microvascular endothelial cells. After 3-5 days, the epithelial cells self-assembled into tubular structures of up to 1 cm, with a lumen lined by a monolayer of polarized epithelial cells at 10 days. In contrast, endothelial cells assembled into tubules with multiple fine branches. We found that a complex pattern of tubular networks of significant length and regular anatomical shape was achieved by molding ECM gels through microfabricated grooved templates.